Method for establishing transgenic removable human skin fibroblast feeder cell line

A technology of fibroblasts and establishment methods, which is applied in the field of establishment of transgene-removed human skin fibroblast feeder cell lines, can solve the problems of feeder cell mixed cell sheet, self-pollution, increased workload and production cost of proliferation ability, and achieve The effect of vigorous splitting ability

Inactive Publication Date: 2020-08-25
SHENZHEN HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the limited proliferative ability of normal human cells cultured in vitro increases the workload and production costs, hindering the clinical applic...

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  • Method for establishing transgenic removable human skin fibroblast feeder cell line
  • Method for establishing transgenic removable human skin fibroblast feeder cell line
  • Method for establishing transgenic removable human skin fibroblast feeder cell line

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Embodiment Construction

[0071] In order to make the above objects, features and advantages of the present invention more comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0072] figure 1 .Flow chart of TERT+TK-D cell creation TERT+TK-D cell creation is divided into two steps: First, human skin fibroblasts (D) were infected with pLenti-TERT-IRES-EGFP-Neo lentivirus, and 800 μg / mLG418 After screening, TERT+EGFP-D cells were obtained. Then, TERT+EGFP-D cell...

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Abstract

The invention discloses a method for establishing a transgenic removable human skin fibroblast feeder cell line. By utilizing a retrovirus-mediated gene transfer way, the production tool introduces areinforced green fluorescent protein gene, a telomerase reverse transcriptase gene and a herpes simplex virus thymidine kinase gene into human skin fibroblasts to establish a fluorescence-labeled immortalization-removable TERT + TK-D human feeder cell line. After being treated by mitomycin C, the established cell line serving as feeder cells is co-cultured with human limbal stem cells, and the culture result is compared with a culture result of 3T3 feeder cells. Transgenic fluorescence-labeled immortalization-removable human skin fibroblast feeder cells are expected to replace the 3T3 cells for corneal regeneration therapy.

Description

technical field [0001] The invention relates to the field of corneal cells, in particular to a method for establishing a transgene-removed human skin fibroblast feeder cell line. Background technique [0002] Limbal stem cells and oral mucosal epithelial cells have been successfully used in ocular surface regenerative medicine to treat severe ocular surface diseases. However, the mouse 3T3 fibroblasts used in the therapy carry the risk of transmitting the murine disease to the patient. Recent reports have shown that human embryonic stem cells co-cultured with mouse feeder cells express the mouse xenoantigen Neu5Gc and can induce an autoimmune response. Therefore, it is necessary to find a safe and effective human-derived feeder cells to replace mouse 3T3 cells. Some recent studies have reported that human dermal fibroblasts have the ability to maintain the growth of corneal limbal and oral mucosa epithelial stem cells and maintain the biological characteristics of epitheli...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/65C12N5/079C12Q1/6851A61L27/38A61L27/36G01N33/58
CPCA61L27/3687A61L27/3813A61L2430/16C12N5/0621C12N5/0623C12N5/0625C12N15/65C12N15/86C12N2500/25C12N2501/01C12N2501/39C12N2501/395C12N2501/999C12N2502/09C12N2533/54C12N2740/15043C12Q1/6851G01N33/582C12Q2531/113C12Q2563/107C12Q2545/101C12Q2565/125
Inventor 黎颖莉李晓霞符敏席蕾
Owner SHENZHEN HOSPITAL OF SOUTHERN MEDICAL UNIV
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